Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
ACS Synth Biol. 2022 Oct 21;11(10):3440-3450. doi: 10.1021/acssynbio.2c00345. Epub 2022 Oct 7.
Engineering of bacterial genomes is a fundamental craft in contemporary biotechnology. The ability to precisely edit chromosomes allows for the development of cells with specific phenotypes for metabolic engineering and for the creation of minimized genomes. Genetic tools are needed to select for cells that underwent editing, and dual-selection markers that enable both positive and negative selection are highly useful. Here, we present an optimized and easy-to-use version of the dual-selection marker and demonstrate how this can be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, can be used to create scarless knockouts across the genome with efficiency above 90%, whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the RNA and protein level, we show that enables advanced genome engineering of both gene translation and transcription by introducing sequence variation in the translation initiation region or by exchanging promoters. Finally, we demonstrate the use of for genome engineering in the industrially relevant probiotic strain Nissle.
细菌基因组工程是当代生物技术的一项基础技术。精确编辑染色体的能力可用于开发具有特定表型的细胞，以进行代谢工程和最小化基因组的构建。需要遗传工具来选择经历编辑的细胞，而能够同时进行正选择和负选择的双选择标记则非常有用。在这里，我们提出了一种经过优化且易于使用的双选择标记版本，并展示了如何有效地在分子生物学中心法则的不同阶段进行工程改造。在 DNA 水平上，该标记可用于在整个基因组中创建无痕敲除，效率超过 90%，而重组基因整合的效率约为 50%。在 RNA 和蛋白质水平上，我们表明该标记通过在翻译起始区引入序列变异或交换启动子，可用于基因翻译和转录的高级基因组工程。最后，我们展示了该标记在工业相关益生菌菌株 Nissle 中的基因组工程中的应用。
